Our early crystal structures showed how iron transporters specifically recognize Fe3+ bound to small molecules such as enterobactin (a siderophore synthesized by Escherichia coli) and citrate. Each transporter has a unique binding pocket for its preferred small molecule. When the correct substrate binds, the transporter undergoes conformational changes that send a signal across the outer membrane and prepare the system for transport. We recently expanded our studies in this area to determine how Neisseria meningitidis binds to human serum transferrin and extracts the iron for import into the bacterial cell. These bacteria require iron for survival and obtain it directly from human proteins. Neisseria have an outer membrane protein, TbpA, and a co-receptor protein, TbpB, which together can extract the iron from a human plasma protein called transferrin. We used a combined approach of X-ray crystallography, electron microscopy, small angle X-ray scattering, biochemistry, and molecular dynamics simulations to elucidate the iron-scavenging mechanism. This was the first atomic resolution structure of a bacterial outer membrane protein bound to its full-length human target protein (Noinaj et al and Buchanan, Nature 2012). We have expanded our focus on transition metal transport to Acinetobacter baumanni zinc uptake. A. baumannii is a hospital-acquired infection demonstrating multidrug resistance. It is of great interest to the NIH clinical center. Zinc correlates with virulence in A. baumannii and there are three putative TonB-dependent zinc transporters in this bacterium. When deprived of zinc, A. baumannii becomes much more sensitive to existing antibiotics, so inhibition of zinc uptake may lead to novel therapies against this Gram-negative bacterium. We just solved the structure of an A. baumannii zinc transporter with zinc bound. We are using the structure to perform in silico small molecule drug screening and to investigate the zinc transport pathway. A manuscript describing this work is in preparation. Interestingly, for all of these transition metal transporters, how the metal gets into the periplasm is not well understood. We know that transport involves an inner membrane protein complex (TonB-ExbB-ExbD) and energy in the form of protonmotive force. We are currently working to provide structural data on the transport process. In our search for novel antimicrobial therapies, we extended our work on small-molecule transporters to ask how proteins are ferried across the outer membrane. Some of the iron transporters that we study also facilitate the uptake of large protein toxins called colicins. For example, we determined the structure of an outer membrane iron transporter from Yersinia pestis (which causes plague) that is required for virulence. We also determined the structure of a colicin, called pesticin, which uses this transporter to cross the outer membrane. The two structures showed us how to engineer a novel antibiotic that is the first example of phage therapy for any Gram-negative bacterium, and our antibiotic was demonstrated to be effective on a number of clinical isolates (Lukacik et al and Buchanan, PNAS 2012). Guided by this success, we will continue this type of protein engineering for other bacterial pathogens. Another hospital-acquired infection of great importance to the NIH clinical center is Klebsiella pneumoniae. This bacterium exhibits multidrug resistance and some strains have shown hypervirulence. In an effort to identify new ways to combat infection, we are collaborating with Susan Gottesman, NCI, to investigate proteins involved in regulation of capsule. K. pneumoniae can escape immune detection and prevent penetration of antibiotics with its thick capsule layer that surrounds the outer membrane. Our hypothesis is that down-regulation of capsule synthesis might make K. pneumoniae more sensitive to available antibiotics, and thus more treatable than is currently the case. Structural and functional experiments on this system are in progress. Finally, in addition to studying outer membrane transporters, we are interested in any outer membrane protein complexes that are essential for bacterial growth, since these proteins may make good vaccine and drug targets. One such complex is LptDE, which puts newly synthesized lipopolysaccharide (LPS) into the outer membrane. In most Gram-negative bacteria, this complex is essential for viability. We recently crystallized LptDE from three different bacterial pathogens and structure solution for all three is underway. We will use these structures to develop new antimicrobial reagents.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
Type
DUNS #
City
State
Country
Zip Code
Ghequire, Maarten G K; Kemland, Lieselore; Anoz-Carbonell, Ernesto et al. (2017) A Natural Chimeric Pseudomonas Bacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter. MBio 8:
Abeykoon, Amila H; Noinaj, Nicholas; Choi, Bok-Eum et al. (2016) Structural Insights into Substrate Recognition and Catalysis in OmpB by Protein Lysine Methyltransferases from Rickettsia. J Biol Chem :
Strickland, Madeleine; Stanley, Ann Marie; Wang, Guangshun et al. (2016) Structure of the NPr:EINNtr Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems. Structure 24:2127-2137
Noinaj, Nicholas; Mayclin, Stephen; Stanley, Ann M et al. (2016) From Constructs to Crystals - Towards Structure Determination of ?-barrel Outer Membrane Proteins. J Vis Exp :
Botos, Istvan; Majdalani, Nadim; Mayclin, Stephen J et al. (2016) Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens. Structure 24:965-976
Celia, Hervé; Noinaj, Nicholas; Zakharov, Stanislav D et al. (2016) Structural insight into the role of the Ton complex in energy transduction. Nature 538:60-65
Cash, Devin R; Noinaj, Nicholas; Buchanan, Susan K et al. (2015) Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae. Infect Immun 83:4438-49
Noinaj, Nicholas; Buchanan, Susan K (2014) FhaC takes a bow to FHA in the two-partner do-si-do. Mol Microbiol 92:1155-8
Noinaj, Nicholas; Buchanan, Susan K (2014) Structural insights into the transport of small molecules across membranes. Curr Opin Struct Biol 27:8-15
Noinaj, Nicholas; Cornelissen, Cynthia Nau; Buchanan, Susan K (2013) Structural insight into the lactoferrin receptors from pathogenic Neisseria. J Struct Biol 184:83-92

Showing the most recent 10 out of 16 publications